The goal of this proposal is to reveal the mechanisms by which pluripotency transcription factors gain access to developmentally silenced genes, open the chromatin, and thereby initiate self-sustaining regulatory networks. Only 1/100 to 1/10,000 of somatic cells in which the Oct4, Sox2, Klf4, and c-Myc factors are expressed can be reprogrammed to pluripotency, even with the most efficient methods of protein, mRNA, or gene delivery and despite the demonstrable co-expression of the factors in the initial cells. My laboratory has shown that FoxA transcription factors have the intrinsic biochemical capacity to bind target sites in the compacted chromatin of silent genes. This creates local hypersensitivity that facilitates binding by other transcription factors and gene activation. We further found that FoxA """"""""pioneer factors"""""""" contain distinct protein domains that modulate the ability of the factor to move in chromatin and to create hypersensitivity, and we found chromatin modification states that restrict FoxA engagement. Based on the profound ability of Oct4, Sox2, Klf4, and c-Myc to activate the silent pluripotency network in differentiated cells, along with role of Nanog in the process, we hypothesize that one or more of such proteins has """"""""pioneer"""""""" activity, along with relevant domains that affect chromatin functions;that such domains may provide sensitivity to resistant chromatin states;and that knowledge of such will allow us to engineer pluripotency factors to more efficiently initiate the reprogramming process. Based on our studies of Fox proteins, we are well positioned to unveil the mechanistic basis for pluripotency factor function and to determine how to enhance it, with these Aims: 1) To assess the initial chromatin engagement and opening properties for the five human pluripotency factors on natural human target sequences reconstituted into purified chromatin templates in vitro, in comparison to the factors'initial engagement on the same chromatin sites in vivo (with Plath and Lowry labs). 2) To map protein domains crucial for chromatin engagement and opening, and the mechanism of action of relevant domains (with Plath lab). 3) To determine what modifications of chromatin can limit the engagement or chromatin opening by the pluripotency factors, and how such may be overcome by modifying the appropriate domains of the factors (with Lowry and Smale labs). Each of these aims involves direct interactions and collaborations with other members of the P01 proposal. Insights and approaches from these studies will be applicable to diverse types of cell and therapeutic contexts for reprogramming. There will be no human or animal experimentation with the proposed work.